CN106047713A - Talaromyces pinophilum strain Li-93 and application thereof - Google Patents
Talaromyces pinophilum strain Li-93 and application thereof Download PDFInfo
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Abstract
The invention provides a Talaromyces pinophilum strain Li-93 which is preserved in China General Microbiological Culture Collection Center, with an accession number of CGMCC No. 11765. The invention also provides a product used for preparing glycyrrhetinic acid monoglucuronide (GAMG), and the active component of the product is Talaromyces pinophilum Li-93. The invention also provides a preparation method for GAMG. When used for preparation of GAMG, the product and method have the characteristics of a single product and no by-product and allow the yield of GAMG prepared under conventional conditions to be about 96%, higher than the yield of GAMG in the prior art. Thus, the invention provides the simple and effective product and method for preparation of GAMG.
Description
Technical field
The invention belongs to biological technical field, be specifically related to one strain produce beta-glucuronidase enzyme addicted to loose basket bacteria strain
And application.
Background technology
Glycyrrhizic acid (Glycyrrhizin, GL) be Radix Glycyrrhizae (Glycyrrhiza uralensis) principle active component it
One, be a kind of triterpene saponin componds, can have an antiinflammatory, anticancer as natural sweeting agent, antitumor, hepatoprotective and
Strengthen Culture in vitro effect, additionally there is good whitening function, in conjunction with the function of its antiinflammatory, can play skin moistening and
Remove the multiple efficacies such as free radical, be therefore also widely used for cosmetic field.But owing to glycyrrhizic acid polarity is relatively strong, it is difficult to
Enter intracellular performance pharmacological action through cell membrane, not only limit the performance of its function, also result in the waste of medicine.Single Fructus Vitis viniferae
Alditol acidic group enoxolone (Glycyrrhetinic acid monoglucuronide, GAMG) is glycyrrhizic acid outermost glucosides
After bond cleavage solution, the product that hydrolysis generates after removing a part glucuronic acid, have because of it while retaining glycyrrhizic acid effect
Polarity is moderate, it is easy to through cell membrane, the advantages such as bioavailability is high, and has higher sugariness, more preferable biological safety
It it is considered as the good succedaneum of glycyrrhizic acid.
Whole-cell catalytic refers to utilize complete living organism to carry out substrate catalysis as catalyst and convert, and its essence is
Utilize the enzyme in cell to be catalyzed, but compared to enzyme catalysis method, eliminate the extraction and purification process of enzyme, prepare simpler
Single, production cost is lower.This area produces the method for GAMG to be had a variety of, wherein utilizes microbial cell catalytic substrate to convert and is
A kind of effective means.Application for a patent for invention 201410088328.3 utilizes the strain penicillium purpurogenum being separated to
Penicilliumpurpurogenum Li-3, can convert production GAMG after immobilized cell processes continuously, use and do not exist together
Reason condition, the yield preparing GAMG is respectively 8.5%, 16.9% and 34%.But, the lowest yield is to subsequent extracted and pure
Change GAMG and produce burden, and cause the waste of substrate, it is difficult to meet low cost, the production requirement of scale.Therefore, this area
Need screening badly a kind of for preparing GAMG and the high new strains of productivity.
Summary of the invention
The present invention is based on the above-mentioned deficiency in this area and demand, it is provided that a strain separating obtained addicted to loose basket bacteria strain
(Talaromycespinophilum) Li-93, this bacterial strain can be used for preparing GAMG, and GAMG productivity is higher.
Technical scheme is as follows:
One strain is addicted to loose basket bacterium (Talaromyces pinophilum) bacterial strain Li-93, and its deposit number is: CGMCC
No.11765。
For preparing the goods of GAMG, it is characterised in that the active component bag of described goods
Include described addicted to loose basket bacteria strain Li-93.
For preparing the goods of GAMG, it is characterised in that the active component of described goods is
Described addicted to loose basket bacteria strain Li-93.
Described goods also include the conventional ingredient for preparing GAMG and/or for cultivating
The medium component addicted to loose basket bacteria strain Li-93 stated.
The preparation method of a kind of goods preparing GAMG, it is characterised in that described in employing
Addicted to loose basket bacteria strain Li-93 as one of active component or active component of preparing described goods；And/or
It is placed with described addicted to loose basket bacterium in indicating the packing box preparing GAMG purposes
Bacterial strain Li-93.
A kind of method preparing GAMG, it is characterised in that at the single glucal acidic group of preparation
Add during enoxolone and/or described in using addicted to loose basket bacteria strain Li-93.
For preparing the microbial inoculum of GAMG, it is characterised in that the active component bag of described microbial inoculum
Include described addicted to loose basket bacteria strain Li-93.
For preparing the microbial inoculum of GAMG, it is characterised in that the active component of described microbial inoculum is
Described addicted to loose basket bacteria strain Li-93.
Described microbial inoculum, also includes the conventional ingredient for preparing microbial inoculum.
The described application addicted to loose basket bacteria strain Li-93 in terms of preparing GAMG.
The present invention is separated to a strain filamentous fungi, through ITS order-checking, sequence alignment, bacterium from Xinjiang licorice planting ground soil
Fall and thalli morphology identify, determine this bacterial strain be a strain addicted to loose basket bacterium (Talaromyces pinophilus), it is ordered
Entitled addicted to loose basket bacterium Li-93.
The present invention to above-mentioned addicted to loose basket bacterium Li-93 in the effect side preparing GAMG (GAMG)
Face is verified, finds: the orientable GAMG that glycyrrhizic acid or its salt is converted into of this bacterium, and the generation of no coupling product GA,
Preparing GAMG yield under normal condition is about 96%, higher than state of the art.
As can be seen here, the present invention provide addicted to loose basket bacterium (Talaromyces pinophilus), bacterial strain Li-93 can be relatively
For preparing GAMG efficiently, and this bacteria growing condition is less demanding, it is easy, with low cost to obtain, prepare GAMG for this area and carry
A kind of simple and effective product and method are supplied.
Described having sent preservation addicted to loose basket bacterium (Talaromyces pinophilum) bacterial strain Li-93, its preservation information is such as
Under:
Culture presevation title: Li-93
Preserving number: CGMCC No.11765
Classification And Nomenclature: addicted to loose basket bacterium
Latin name: Talaromyces pinophilum
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on November 30th, 2015
Accompanying drawing explanation
Fig. 1 is addicted to loose basket bacterium (Talaromyces pinophilum) Li-93 optical microscope；
Fig. 2 is to convert monoammonium glycyrrhizinate addicted to loose basket bacterium to generate the efficient of single glucuronic acid base glycyrrhetic acid (GAMG)
Liquid-phase chromatographic analysis figure, 1: the standard substance of enoxolone (GA)；The standard substance of 2:GAMG；3: before conversion；After 4 convert.
Detailed description of the invention:
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following example are merely to illustrate this
Invent not for limiting the scope of the present invention.Experimental technique used in following embodiment if no special instructions, is often
Rule method.Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Licorice planting ground, the Xinjiang soil collecting used in embodiment 1 is from Emin County, Xinjiang, China Tacheng licorice planting ground.
The purity of the monoammonium glycyrrhizinate used in embodiment is 75%, is purchased from Xinjiang Tianshan pharmaceutical factory.
The culture medium used in following example is described below:
Screening flat board: monoammonium glycyrrhizinate 3g/L, NaNO33g/L, K2HPO41g/L, KCl 1g/L, MgSO4·7H2O
0.5g/L, Fe2SO4·7H2O 0.01g/L, pH 5.0, agar 30g/L.
Cha Shi yeast extract agar culture medium (CYA): NaNO33g/L, K2HPO41g/L, MgSO4·7H2O 0.5g/L, KCl
0.5g/L, FeSO4 7H2O 0.01g/L, sucrose 20g/L, yeast powder extract 5g/L, agar 20g/L, natural pH.
Wort agar culture medium (MEA): Fructus Hordei Germinatus extract 20g/L, glucose 20g/L, polyprotein peptone 1g/L, agar
20g/L, natural pH.
Oat-agar cultures base (OA) preparation method is as follows:
Take 30g Herba bromi japonici and add water to 1000mL, boil filtered through gauze after 1 hour, take filtrate and add water and complement to 1000mL, add
20g agar, 121 DEG C of high temperature sterilize 20min.
Potato glucose liquid (Gu) body culture medium preparation method is as follows:
Weighing peeled potatoes 200g, add water 800mL, boils about 30 minutes, filtered through gauze, adds 20g in filtrate
Glucose, dissolve after supply water to 1000mL, if needing to make solid medium, add 20g agar the most again, natural
PH, 121 DEG C of high temperature sterilize 15min.
Secondary seed medium: peeled potatoes 200g, add water 800mL, boils about 30 minutes, filtered through gauze, takes filter
Liquid supplies water to being taken out 250mL after 1000mL, adds 8g glucose, after dissolving, adds water to 1000mL, natural pH, 121 DEG C
High temperature sterilize 15min.
The preparation method of fermentation medium is as follows:
Taking peeled potatoes 200g, add water 800mL, boils about 30 minutes, filtered through gauze, takes filtrate and supplies water extremely
It is taken out 200mL after 1000mL, adds water to 5.0,121 DEG C of high temperature sterilize 15min of 1000mL, pH.
In the present invention, the productivity of GAMG is defined as:
Embodiment 1: addicted to screening and the qualification of loose basket bacterium Li-93
Gathering licorice planting ground, Xinjiang soil 10g, be placed in 100mL aseptic 0.8%NaCl solution, fully vibrate 10min,
Aseptic filter paper filters, and takes filtrate and dilutes 10 respectively1、102、103、104Times, each diluent is coated on screening flat board, in 28
DEG C cultivate.Every 24h observes whether flat board grows the bacterium colony made new advances, and separation of being rule further by newly long bacterium colony is to obtain purebred bacterium
Strain.Therefrom it is separated to a strain filamentous fungi, names Li-93.
The ITS sequence of this bacterial strain Li-93 is checked order, shown in result as SEQ ID No.1.Sequence is carried out BALST
Comparison, finds this bacterium and Talaromyces pinophilus NBRC100533T、Talaromyces pinophilus NRRL
The TIS sequence of 58691 and Talaromyces pinophilum NRRL 6420 has the homology of more than 99%.
This bacterium spore is connected to Cha Shi yeast extract agar culture medium, wort agar culture medium, oat-agar cultures respectively
Base, cultivates 5-7 days, observes this bacterium and grow the colonial morphology of formation in various culture medium for 30 DEG C.
Result shows: after cultivating 7 days in 28 DEG C in Cha Shi yeast extract agar culture medium, colony diameter is about 30mm, bacterium
Silk is in velvet-like, yellow, and quality is fine and close, and colony edge is white, forms a circle groove, and enclose with this at distance center point about 7mm
Groove is starting point, is outwardly formed the radial recessed grain of 1-5 bar, and the back side is orange red.In 28 DEG C of trainings in wort agar culture medium
Supporting after 7 days, colony diameter is about 40mm, and mycelia is that heavy fleece shape, quality be of a relatively loose, color is followed successively by white, Huang from outside to inside
Normal complexion bottle green, the circular protrusions of intermediate formation about 12mm diameter, the back side is orange red.In 28 DEG C on oat-agar cultures base
After cultivating 7 days, colony diameter is about 40mm, and mycelia is cotton-shaped, and quality is loosened, and edge be white, and centre is bottle green, the back side without
Color or local are in greenish orange color.
Basis of microscopic observation: mycelia has barrier film, top to have the conidial head of broom shape, and upright conidiophore has
The raw stigma of two-wheel, symmetric form, conidium is that string is arranged in stigma top, spherical in shape or subsphaeroidal, as shown in Figure 1.
According to result above, evaluation and screening obtained strains is addicted to loose basket bacterium (Talaromyces pinophilus), quilt
Named Li-93.This bacterial strain Li-93 send preservation, and the bacterial strain being claimed as the present invention simultaneously, and its preserving number is
CGMCC No.11765。
Embodiment 2, use and of the present invention prepare GAMG addicted to loose basket bacteria strain Li-93
To be connected on inclined-plane potato glucose solid medium addicted to loose basket bacterium Li-93, cultivate 3 days in 30 DEG C.Take tiltedly
Spore inoculating on face on potato dextrose broth, in 30 DEG C, cultivate 48h on the shaking table of 200rpm, then with
The inoculum concentration of volume ratio 5% is connected in secondary seed medium, 30 DEG C, cultivate 24h on the shaking table of 200rpm, obtains secondary seed
Liquid.By secondary seed solution, it is connected in fermentation medium with the inoculum concentration of volume ratio 10%, 30 DEG C, cultivate 12h on 200rpm shaking table
After, be added thereto to monoammonium glycyrrhizinate and make its final concentration reach 5g/L, in 30 DEG C, 200rpm shake bed reaction.Every 6h sampling,
Use consumption and the growing amount of GAMG of high performance liquid chromatography detection monoammonium glycyrrhizinate, after result display 24h, glycyrrhizic acid list
Ammonium salt converts complete, and GAMG is again without substantially increasing, as shown in Figure 2.Being computed, the productivity of GAMG reaches 96.31%.
Embodiment 3, the goods for preparing GAMG of the present invention
The present embodiment provides a kind of goods for preparing GAMG, the active component of these goods
Including embodiment 1 isolated addicted to loose basket bacteria strain Li-93；And/or,
The active component of these goods be described in embodiment 1 addicted to loose basket bacteria strain Li-93.
Based on being used for preparing the goods of GAMG described in the present embodiment, also include for preparing
The conventional ingredient of GAMG and/or for cultivating the described cultivation addicted to loose basket bacteria strain Li-93
Based component.
The present embodiment also provides for the preparation method of said products, comprise the steps: to use described in embodiment 1 addicted to pine basket
Shape bacteria strain Li-93 is as one of active component or active component of preparing described goods；And/or
Be placed with in indicating the packing box preparing GAMG purposes described in embodiment 1 addicted to pine
Basket bacteria strain Li-93.
Embodiment 4, the microbial inoculum for preparing GAMG of the present invention
The present embodiment provides a kind of microbial inoculum for preparing GAMG, and feature is, described microbial inoculum
Active component include described in embodiment 1 addicted to loose basket bacteria strain Li-93；And/or
The active component of described microbial inoculum be described in embodiment 1 addicted to loose basket bacteria strain Li-93.
Described microbial inoculum also includes the conventional ingredient for preparing microbial inoculum.
Claims (10)
1. a strain is addicted to loose basket bacterium (Talaromyces pinophilum) bacterial strain Li-93, and its deposit number is: CGMCC No:
11765。
2. for preparing the goods of GAMG, it is characterised in that the active component of described goods includes
Described in claim 1 addicted to loose basket bacteria strain Li-93.
3. for preparing the goods of GAMG, it is characterised in that the active component of described goods is power
Profit require described in 1 addicted to loose basket bacteria strain Li-93.
4. according to the goods described in Claims 2 or 3, it is characterised in that described goods also include for preparing single glucal
The conventional ingredient of acidic group enoxolone and/or for cultivating the described medium component addicted to loose basket bacteria strain Li-93.
5. the preparation method of the goods preparing GAMG, it is characterised in that use claim 1
Described addicted to loose basket bacteria strain Li-93 as one of active component or active component of preparing described goods；And/or,
Be placed with in indicating the packing box preparing GAMG purposes described in claim 1 addicted to pine basket
Shape bacteria strain Li-93.
6. the method preparing GAMG, it is characterised in that sweet at the single glucal acidic group of preparation
Grass subacid during add and/or use described in claim 1 addicted to loose basket bacteria strain Li-93.
7. for preparing the microbial inoculum of GAMG, it is characterised in that the active component of described microbial inoculum includes
Described in claim 1 addicted to loose basket bacteria strain Li-93.
8. for preparing the microbial inoculum of GAMG, it is characterised in that the active component of described microbial inoculum is power
Profit require described in 1 addicted to loose basket bacteria strain Li-93.
9. according to the microbial inoculum of claim 7 or 8, it is characterised in that also include the conventional ingredient for preparing microbial inoculum.
10. described in claim 1 addicted to loose basket bacteria strain Li-93 in terms of preparing GAMG should
With.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106591071A (en) * | 2016-12-23 | 2017-04-26 | 江苏耐雀生物工程技术有限公司 | Healthcare baijiu and preparation method thereof |
| CN107267400A (en) * | 2017-06-27 | 2017-10-20 | 河南师范大学 | One plant of basket bacterium and the method using the basket bacteria liquid mycoderm fermenting and producing betanin |
| CN113322192A (en) * | 2021-07-15 | 2021-08-31 | 云南省农业科学院药用植物研究所 | Polygonatum sibiricum endophytic fungi escherichia coli inhibiting agent and preparation method and application thereof |
| CN116555041A (en) * | 2023-02-10 | 2023-08-08 | 江苏农林职业技术学院 | Strawberry biocontrol endophytic fungus JSNL-B124 and application thereof |
| CN117535156A (en) * | 2022-12-01 | 2024-02-09 | 河北省科学院生物研究所 | Brucella CFT-1 and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106591071A (en) * | 2016-12-23 | 2017-04-26 | 江苏耐雀生物工程技术有限公司 | Healthcare baijiu and preparation method thereof |
| CN107267400A (en) * | 2017-06-27 | 2017-10-20 | 河南师范大学 | One plant of basket bacterium and the method using the basket bacteria liquid mycoderm fermenting and producing betanin |
| CN107267400B (en) * | 2017-06-27 | 2020-03-10 | 河南师范大学 | Talaromyces and method for producing betanin by fermenting liquid mycoderm of Talaromyces |
| CN113322192A (en) * | 2021-07-15 | 2021-08-31 | 云南省农业科学院药用植物研究所 | Polygonatum sibiricum endophytic fungi escherichia coli inhibiting agent and preparation method and application thereof |
| CN117535156A (en) * | 2022-12-01 | 2024-02-09 | 河北省科学院生物研究所 | Brucella CFT-1 and application thereof |
| CN117535156B (en) * | 2022-12-01 | 2024-05-17 | 河北省科学院生物研究所 | Brucella CFT-1 and application thereof |
| CN116555041A (en) * | 2023-02-10 | 2023-08-08 | 江苏农林职业技术学院 | Strawberry biocontrol endophytic fungus JSNL-B124 and application thereof |
| CN116555041B (en) * | 2023-02-10 | 2024-03-01 | 江苏农林职业技术学院 | Strawberry biocontrol endophytic fungus JSNL-B124 and application thereof |
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